1. Field of the Invention
The present invention relates to triple hybrid amplicon vector constructs comprising elements from Herpes Simplex virus (HSV), Epstein-Barr virus (EBV) or Adeno-Associated Virus (AAV), and retrovirus. The hybrid amplicon vectors of the present invention are capable of transforming dividing and non-dividing cells into retroviral packaging cells in a single step, which can be mediated in vitro or in vivo. Because the vector system of the present invention can convert cells in vivo to packaging cells, it creates, in vivo, a self-sustained gene delivery system.
2. Related Art
The terms xe2x80x9cgene transferxe2x80x9d and xe2x80x9cgene therapyxe2x80x9d have been used to describe a variety of methods for delivering genetic material to a cell using viral or non-viral based vector systems. Substantial attention has been given to human gene therapy. The transfer of genetic material to a cell may one day become one of the most important forms of medicine. A variety of public and private institutions now participate in research and development related to the use of genetic material in therapeutic applications. Hundreds of human gene transfer protocols are being conducted at any given time with the approval of the Recombinant DNA Advisory Committee (RAC) and the National Institutes of Health (NIH). Most of these protocols focus on therapy, while others involve marking and non-therapeutic applications. The therapeutic protocols are primarily concerned with infectious diseases, monogenic diseases, and cancer. Gene-based therapies are now expanding into fields such as cardiovascular disease, autoimmune disease, and neurodegenerative disease. The availability of an efficient gene delivery and expression system is essential to the success and efficacy of gene-based therapy.
One method of delivering a gene of interest to a target cell of interest is by using a viral-based vector. Techniques for the formation of vectors or virions are generally described in xe2x80x9cWorking Toward Human Gene Therapy,xe2x80x9d Chapter 28 in Recombinant DNA, 2nd Ed., Watson, J. D. et al., eds., New York: Scientific American Books, pp. 567-581 (1992). An overview of viral vectors or virions that have been used in gene therapy can be found in Wilson, J. M., Clin. Exp. Immunol. 107(Suppl. 1):31-32 (1997), as well as Nakanishi, M., Crit. Rev. Therapeu. Drug Carrier Systems 12:263-310 (1995); Robbins, P. D., et al., Trends Biotechnol. 16:35-40 (1998); Zhang, J., et al., Cancer Metastasis Rev. 15:385-401 (1996); and Kramm, C. M., et al., Brain Pathology 5:345-381 (1995). Such vectors may be derived from viruses that contain RNA (Vile, R. G., et al., Br. Med Bull. 51:12-30 (1995)) or DNA (Ali M., et al., Gene Ther. 1:367-384 (1994)).
Specific examples of viral vector systems that have been utilized include: retroviruses (Vile, R. G., supra; U.S. Pat. Nos. 5,741,486 and 5,763,242); adenoviruses (Brody, S. L., et al., Ann. N.Y. Acad. Sci. 716: 90-101 (1994); Heise, C. et al., Nat. Med. 3:639-645 (1997)); adenoviral/retroviral chimeras (Bilbao, G., et al., FASEB J. 11:624-634 (1997); Feng, M., et al., Nat. Biotechnol. 15:866-870 (1997)); adeno-associated viruses (Flotte, T. R. and Carter, B. J., Gene Ther. 2:357-362 (1995); U.S. Pat. No. 5,756,283); herpes simplex virus I or II (Latchman, D. S., Mol. Biotechnol. 2:179-195 (1994); U.S. Pat. No. 5,763,217; Chase, M., et al., Nature Biotechnol. 16:444-448 (1998)); parvovirus (Shaughnessy, E., et al., Semin Oncol. 23:159-171 (1996)); reticuloendotheliosis virus (Donburg, R., Gene Therap. 2:301-310 (1995)). Other viruses that can be used as vectors for gene delivery include poliovirus, papillomavirus, vaccinia virus, lentivirus, as well as hybrid or chimeric vectors incorporating favorable aspects of two or more viruses (Nakanishi, M. Crit. Rev. Therapeu. Drug Carrier Systems 12:263-310 (1995); Zhang, J., e al., Cancer Metastasis Rev. 15:385-401 (1996); Jacoby, D. R., et al., Gene Therapy 4:1281-1283 (1997)). Guidance in the construction of gene therapy vectors and the introduction thereof into affected animals for therapeutic purposes may be obtained in the above-referenced publications, as well as U.S. Pat. Nos. 5,631,236, 5,688,773, 5,691,177, 5,670,488, 5,529,774, 5,601,818, and WO 95/06486.
The viral vectors mentioned above each have advantages and disadvantages. For example, retroviruses have the ability to infect cells and have their genetic material integrated into the host cell with high efficiency. The development of a helper virus free packaging system for retrovirus vectors was a key innovation in the development of this vector system for human gene therapy. Retroviral helper virus free packaging systems generally employ the creation of a stable producer cell line that expresses a selected vector. The relatively small size of the retroviral genome (approximately 11 kb), and the ability to express viral genes without killing cells, allows for the production of a packaging cell line that synthesizes all the proteins required for viral assembly. Producer lines are made by introducing the retroviral vector into such a packaging cell line.
On a down side, however, numerous difficulties with retroviruses have been reported. For example, most retroviral vectors are not capable of gene transfer to postmitotic (nondividing) cells and thus are not applicable to the nervous system because most of the cells in the adult nervous system, especially neurons, are quiescent or postmitotic. In addition, outbreaks of wild-type virus from recombinant virus-producing cell lines have also been reported, with the vector itself causing a disease.
Difficulties have been noted with other viral vectors as well. Adenovirus vectors can only support limited long-term (2 months) gene expression, they appear to be gradually lost from neural cells, and moreover, they can cause both cytopathic effects and an immune response (Le Gal La Salle, G., et al., Science 259:988-990 (1993); Davidson et al., Nat. Genet. 3:219-223 (1993); Yang, Y., et al., J. Virol. 69:2004-2015 (1995)). Adeno-associated virus vectors cause minimal cytopathic effects and can support at least some gene expression for up to 4 months, but gene transfer is inefficient and these vectors can accept only about 4 kb of foreign DNA (Kaplitt, M. G., et al., Nat. Genet. 8:148-154 (1994)).
Vectors based on herpes simplex virus (HSV), and especially HSV-1, have shown promise as potent gene delivery vehicles for several reasons: the virus has a very large genome and thus can accommodate large amounts of foreign DNA (greater than 30 kb), the virus can persist long-term in cells, and can efficiently infect many different cell types, including post-mitotic neural cells (Breakefield, X. O., et al., xe2x80x9cHerpes Simplex Virus Vectors for Tumor Therapy,xe2x80x9d in The Internet Book of Gene Therapy: Cancer Gene Therapeutics, R. E. Sobol and K. J. Scanlon, eds., Appleton and Lange, Stamford, Conn., pp. 41-56 (1995); Glorioso, J. C., et al., xe2x80x9cHerpes Simplex Virus as a Gene-Delivery Vector for the Central Nervous System,xe2x80x9d in Viral Vectors: Gene Therapy and Neuroscience Applications, M. G. Kaplitt and A. D. Loewy, eds., Academic Press, New York, pp. 1-23 (1995)).
Two types of HSV-1 vector systems are known: recombinant and amplicon. Recombinant HSV-1 vectors (Wolfe, J. H. et al., Nat. Genet. 1:379-384 (1992)) are created by inserting genes of interest directly into the 152 kb viral genome, thereby mutating one or more of the approximately 80 viral genes, and concomitantly reducing cytotoxicity.
In contrast, HSV-1 amplicons are bacterial plasmids containing only about 1% of the 152 kb HSV-1 genome. They are packaged into HSV-1 particles (virions) using HSV-1 helper virus. HSV-1 amplicons contain: (i) a transgene cassette with a gene of interest; (ii) sequences that allow plasmid propagation in E. coli, such as the origin of DNA replication colE1 and the ampicillin resistance gene; and (iii) non-coding elements of the HSV-1 genome, in particular an origin of DNA replication (ori) and a DNA cleavage/packaging signal (pac), to support replication and subsequent packaging of the amplicon DNA into virions in the presence of helper functions (Spaete, R. R. and Frenkel, N., Cell 30:295-304 (1982)). HSV amplicon vectors are one of the most versatile, most efficient, and least toxic, and have the largest transgene capacity of the currently available virus vectors. HSV-1 amplicon vectors can support some gene expression for up to one year in non-dividing cells (During, M. J., et al., Science 266:1399-1403 (1994)).
Because HSV-1 encodes many toxic functions, improvements on the amplicon system have been targeted at reducing the risk associated with the helper virus. First, replication-competent HSV-1, initially used as helper virus, was replaced by a temperature-sensitive (ts) mutant of HSV-1 (HSV-1 tsK; Preston, C., J. Virol. 29:257-284 (1979)). This mutant encodes a temperature-sensitive form of the essential HSV-1 infected cell protein (ICP) 4, allowing HSV-1 replication to proceed at 31xc2x0 C., but not at 37xc2x0 C. Amplicons packaged at 31xc2x0 C. in the presence of HSV-1 tsK were successfully used to transfer the E. coli lacZ gene into primary cultures of rat neural cells (Geller, A. I. and Breakefield, X. O., Science 241:1667-1669 (1988)). Because the infection was performed at 37xc2x0 C., the lytic cycle of the HSV-1 tsK helper virus present in the vector stock was blocked and cell damage was limited. Although replication of HSV-1 tsK was inhibited at the restrictive temperature, the expression of other viral genes caused cytopathic effects. Moreover, reversion to wild type (wt) HSV-1 occurred at a relatively high frequency, due to remaining functionality and reversion of the point mutation in tsICP4.
To counter these problems, replication-defective mutants of HSV-1 were then used as helper viruses (Geller, A. I. et al., Proc. Natl. Acad. Sci. USA 87:8950-8954 (1990); Lim, F., et al., Bio Techniques 20:458-469 (1996)). These mutants carry deletions in genes that are essential for virus replication, but they can support amplicon packaging in cells that complement the missing functions. However, many problems associated with the presence of helper virus in amplicon stocks still remained, including: (i) pronounced cytopathic effects and immune responses caused by gene expression from the helper virus; (ii) interactions between the helper virus and endogenous viruses; (iii) reversion of the helper virus to wt HSV-1; and (iv) disregulation of transgene expression by virus proteins.
Many of these problems have been overcome by the more recent development of a packaging system for herpes virus vectors that was free of helper virus (Fraefel, C., et al., J. Virol. 70:7190-7197 (1996); International Patent Publication WO 97/05263, published Feb. 13, 1997)). This system utilizes transient co-transfection of amplicon DNA with a set of five cosmids that overlap and represent the entire HSV-1 genome, but which are mutated to delete the DNA cleavage/packaging (pac) signals. Cunningham, C. and Davison, A. J., Virology 197:116-124 (1993), had demonstrated previously that after transfection into cells, an overlapping HSV-1 cosmid set can produce infectious virus progeny. By deleting the pac signals and making a pac-minus helper virus genome, HSV-1 genomes that are potentially reconstituted from the cosmids via homologous recombination, are not packageable, but can still provide all the helper functions required for the replication and packaging of the co-transfected amplicon DNA. The resulting vector stocks are, therefore, virtually free of detectable helper virus and have titers of 106-107 tu./ml of culture medium. Because of minimal sequence homology between the cosmids and the amplicon DNA (oriS0.2-1 kb), the formation of a packageable and replication-competent HSV-1 genome is possible but requires 6 recombination events, and is therefore very rare.
Amplicon vector stocks, produced by using the helper virus-free packaging system, can efficiently transduce many different cell types, including neural cells and hepatocytes in culture and in vivo, while causing minimal to no cytopathic effects (Fraefel, C., et al., J. Virol. 70:7190-7197 (1996); Fraefel, C., et al., Mol. Med. 3:813-825 (1997); Fraefel, C., et al., xe2x80x9cHSV-1 Ampliconxe2x80x9d in Gene Therapy for Neurological Disorders and Brain Tumors, E. A. Chiocca and X. O. Breakefield, eds., Humana Press, Totowa, pp. 63-82 (1998); Johnston, K. M., et al., Hum. Gene Ther. 8:359-370 (1997); Aboody-Guterman, K. S., et al., Neuroreport 8:3801-3808 (1997)). Even more recently, helper virus-free packaging has also been achieved using an oversized pac minus HSV genome, defective in an essential gene encoding ICP27, cloned into a BAC plasmid (Saeki, Y., et al., Hum Gene Ther 9:2787-2794 (1998).
One of the main objectives of gene therapy is to achieve stable genetic modification of target cells. This means that their progeny, or themselves in the case of non-dividing cells, should retain and express the newly introduced genetic material until the end of their lifespan, ideally in a regulated manner. This principle is equally valid when transgenes are introduced to correct genetic deficiencies or for treatment of non-hereditary diseases. The viral vector systems discussed above can achieve retention of the transgene through different mechanisms. For example, retrovirus and AAV vectors can integrate genes into the genome of infected cells, while EBV-derived vectors are maintained by episomal replication.
Moloney murine leukemia virus (MoMLV) derived retrovirus vectors are among the most commonly used vectors in gene therapy because of their ability to stably integrate transgenes in the genome of target cells, as well as their relative safety, simplicity, and easy production. However, their use for direct gene delivery in vivo has been limited due to several factors which result in low efficiency of gene transfer. These properties include: low titer, inability to infect non-dividing cells, limited tropism, and relatively short half-life. Although improvements have been made to address some of these issues, MoMLV-based retrovirus vectors are still mostly used for ex vivo protocols. These strategies involve removal of the target cells from the experimental subject or use of donor cells, genetic modification in culture by infection with retrovirus vectors carrying the transgene of interest, selection and characterization of transduced cells, and implantation of these cells in vivo.
An alternative to the direct injection of retroviral particles for in vivo gene delivery is the injection of retrovirus producer cells resulting in the local production of retrovirus vectors. This strategy has been used with therapeutic success in the treatment of experimental brain tumors (Culver, K. W. et al., Science 256:1550-1552 (1992)). In the clinical setting, however, the retrovirus packaging cells remain localized near the injection site, resulting in a very low efficiency of gene delivery to tumor cells, limited to areas in the immediate vicinity of the packaging cells (Ram, Z., et al., Nature Med. 182:1354-1361 (1997)). Further, the fact that the retrovirus packaging cells used are derived from mouse fibroblasts has several disadvantages and potential risks (Isacson, O. and X. O. Breakefield, Nature Med. 3:964-969 (1997)).
First, the retrovirus particles produced are extremely sensitive to inactivation by human serum via complement activation. This occurs because non-primate packaging cells add a Gal(xcex11-3)galactosyl group to the retroviral envelope and human serum has pre-existing antibodies against that sugar group (Rother, R. P., et al., J. Experimen. Med. 182:1345-1355 (1995); Takeuchi, Y., et al., Nature 379:85-88 (1996)). Second, the fact that these cells are mouse fibroblasts presents two difficulties: first, that fibroblasts transplanted into a brain tumor remain largely at the injection site and do not distribute throughout the tumor (Tamiya, T., et al., Gene Ther. 2:531-538 (1995); Tamura, M., et al., Human Gene Ther. 8:381-391 (1997)); second, their xenogeneic origin is likely to exacerbate an immune response even in immune privileged regions such as the central nervous system (CNS), thus limiting their survival time. A third consideration is safety since murine cells carry in their genome a large variety of MLV-like genomes which can be packaged by type C Gag proteins (Chakraborty, A. K., et al., Cancer Gene Ther. 1:113-118 (1994); Hatzoglou, M. et al., Human Gene Ther. 1:385-397 (1990); Scadden, D. T., et al., J. Virol. 64:424-427 (1990)) leading to recombination events which result in the production of replication-competent retroviruses (RCR).
Several packaging cell lines derived from human cells have been developed which could be used instead of the murine versions. These human retrovirus packaging cell lines package fewer endogenous sequences (Patience, C., et al., J. Virol. 72:2671-2676 (1998)), which reduce the probability of generating RCRs, and the virus particles are resistant to inactivation by human serum (Cossett, F. L., et al., J. Virol. 69:7430-7436 (1995)). However, there is also an increased risk for tumor formation since most of these cell lines are derived from transformed cells or human tumors. Since all stable retrovirus packaging cell lines available are derived from fibroblasts, fibrosarcomas or kidney cells, their migratory properties, for example, in the central nervous system (CNS), are very limited, while the target tumor cells present extensive migratory patterns (Pederson, P. H., et al., Cancer Res. 53:5158-5165(1993); Tamura, M. K., et al., Hum. Gene Ther. 8:381-391 (1997).
To increase the efficiency of gene delivery by retrovirus packaging cells, it will be necessary to generate new packaging cell lines derived from cells of different origins than fibroblasts or human kidney cells, preferentially primary cells, which by their migratory or tissue targetting properties will gain access to tumor cells or other target cell populations (Aboody-Guterman, K. S., xe2x80x9cNeural stem cells migrate throughout and express foreign genes within experimental gliomasxe2x80x94a potential gene therapy approach to brain tumorsxe2x80x9d (submitted for publication, 1999); Brown, A. B. et al., xe2x80x9cVascular targeting of therapeutic cells to tumorsxe2x80x9d (in preparation,1999); Lal, B., et al., Proc. Natl. Acad. Sci. USA 91:9695-9699 (1994)). However, generation of retrovirus packaging cell lines is usually an inefficient, cumbersome work that takes, at least, several months to complete.
Several transient transfection systems that produce high titer retrovirus vector stocks in a very short period of time have been developed (Landau, N. R., and Littman, D. R., J. Virol. 66:5110-5113(1992); Naviaux, R. K., et al., J. Virol. 70:5701-5705 (1996); Soneoka, Y., et al., Nucleic Acids Res. 23:628-633 (1995)). Noguiez-Hellin et al. generated retrovirus producing cells in situ by transfection with a plasmid carrying all necessary functions for retrovirus packaging and vector generation (Noguiez-Hellin, P., et al., Proc. Natl. Acad. Sci. USA 93:4175-4180 (1996)). This group showed that the transgene could be generated in culture, but to a lesser extent on a tumor model in vivo, probably due to low transfection efficiency with the plasmid Although these systems produce high retrovirus titers, they are limited by varying efficiencies of transfection between cell lines and their transient nature.
Other systems for producing retroviral vectors take advantage of the efficient gene delivery and expression mediated by other types of viral vectors, such as those derived from herpes simplex virus (HSV) (Savard, N., et al., J. Virol. 71:4111-4117 (1997)), Semliki Forest virus (SFV) (Li, K. J. and H. Garoff, Proc. Natl. Acad. Sci. USA 93:11658-11663 (1996)), and adenovirus (Duisit, G., et al., Human Gene Ther. 10:189-200 (1999); Feng, M., et al., Nature Biotechnol. 15:866-870 (1997); Lin, X., Gene Ther. 5:1251-1258 (1998); Yoshida, Y., et al., Biochem. Biophys. Res. Comm. 232:379-382 (1997)). These systems utilize two or three different vectors to introduce the retroviral vector element and packaging functions, depending on whether the gag-pol and env genes are encoded in the same vector (Feng, M., et al., Nature Biotechnol. 15:866-870 (1997); Lin, X., Gene Ther. 5:1251-1258 (1998); Savard, N., et al., J. Virol. 71:4111-4117 (1997)), or the env gene is delivered by a separate vector (Duisit, G., et al., Human Gene Ther. 10:189-200 (1999); Li, K. J., and H. Garoff, supra; Lin, X., supra; Yoshida, Y., et al., Biochem. Biophys. Res. Comm. 232:379-382 (1997)). Although, one of these adenovirus/retrovirus chimeric systems has been shown to extend the duration of transgene expression in tumors in vivo when compared to a recombinant adenovirus vector (Feng, M., et al., Nature Biotechnol. 15:866-870 (1997)), all of these viral-based systems result in transient production of retrovirus vectors.
As discussed earlier, HSV amplicons are plasmid-based vectors that, in addition to a transgene of interest and corresponding expression elements, only need two non-coding HSV sequences, the origin of DNA replication (ori S) and a packaging signal (xcex1 sequence), to be packaged in HSV virions in the presence of helper functions (Spaete, R. R. and N. Frenkel, Cell 30:295-304 (1982)). These virions can infect a wide range of dividing and non-dividing cells, and with the development of the HSV helper virus-free packaging systems, discussed above, have essentially no toxicity. HSV virions package about 150 kb of DNA. The amplicon DNA is packaged as a concatamer approximately that size, containing multiple copies of the plasmid repeated in tandem, due to a rolling circle mode of viral DNA replication. This presents the advantages that a single virion can transduce a cell with multiple copies of a transgene and that these vectors can carry large genes and regulatory regions (for review, see, Fraefel, C., et al., Gene Therapy for Neurological Disorders and Brain Tumors, Humana Press, Totowa, N.J., pp. 63-82 (1998). However, one major limitation of HSV amplicon vectors has been the loss of amplicon DNA from the host cell nucleus over time, and therefore of gene expression. This is especially true in dividing cells (Johnston, K. M., et al., Hum. Gene Ther. 8:359-370 (1997)).
Recently, two different hybrid amplicon systems have been developed that incorporate elements from other viruses that serve to increase retention of the amplicon DNA and extend transgene expression: The first is an HSV/EBV hybrid amplicon, which includes two EBV elements in its backbone:(1) the latent origin of DNA replication (oriP); and (2) the gene encoding the Epstein-Barr nuclear antigen (EBNA-1), which supports nuclear replication of the amplicon DNA in dividing cells (Wang, S. and J. M. Vos, J. Virol. 70:8422-8430 (1996)). The second is an HSV/AAV hybrid amplicon which incorporates the AAV ITR element and rep gene. These elements have the potential to mediate replicative amplification and chromosomal integration of the transgene cassette (Johnston, K. M., et al., Hum. Gene Ther. 8:359-370 (1997)). This principle of incorporating these AAV elements to achieve chromosomal integration of transgenes with viruses that normally do not possess this property has also been used with baculovirus and adenovirus (Palombo, F., et al., J. Virol. 72:5025-5034 (1998); Recchia, A., et al., Proc. Natl. Acad. Sci. USA 96:2615-2620 (1999)).
Clearly, there is a need in the art for additional and more efficient hybrid amplicon vector systems that are capable of generating stable retrovirus packaging cell lines, in order to stably deliver a transgene, in vitro or in vivo, to a large number and type of dividing or non-dividing cells.
In the present invention, HSV amplicons, in combination with elements from other viruses, are utilized to generate retrovirus packaging cells. More specifically, the present invention relates to the development and characterization of a gene delivery system based on an HSV/EBV or an HSV/AAV hybrid amplicon vector, each of which have been modified to contain retroviral packaging functions and a retrovirus vector cassette. The ability of these hybrid amplicon vectors to induce retrovirus vector production was assessed in a number of different cells, as well as in vivo in a nude mouse model. High titer recombinant retrovirus vectors were obtained both by transfection and infection of different cell lines. These hybrid amplicon vectors can also mediate cumulative transgene delivery in cell populations starting from a small fraction of amplicon-infected cells.
Using current technology, the generation of packaging cell lines is a process which requires several rounds of transfection, selection, and screening. Due to its time consuming nature, most of the cell lines that have been generated have very limited biological interest beyond packaging recombinant retrovirus vectors. Most of the cells are derived from mouse fibroblasts and more recently human cell lines. The vector system of the present invention will be able to shift the burden of work from generating packaging cell lines per se, to using the biologic properties of different cell types for specific applications. Moreover, the generation of packaging cells in vivo will allow direct access to a variety of endogenous cells.
One example of how the vector systems of the present invention will improve the art is in the use of retroviral vectors for cancer gene therapy, which is currently in human clinical trials. The rationale behind these trials is that the retrovirus vectors generated by a packaging cell line, which is injected into a tumor, will infect neighboring dividing tumor cells, rendering them more sensitive to a prodrug, radiation, or conventional chemotherapy. In brain tumors, namely glioblastomas, the main tumor mass is frequently accessible to the neurosurgeon for removal. However, death in most cases results from tumor recurrences which develop close to the original tumor mass, as well as in other places in the central nervous system, sometimes as far away as the opposite hemisphere from where the initial tumor mass was located. This is due to the extraordinary ability that these tumor cells have to migrate and spread in the adult brain. Currently used packaging cell lines derived from rodent fibroblasts, which have been used for brain tumor therapy, remain at the injection site, and some studies have shown that encapsulation of the grafted cells can occur. This suggests that fibroblasts which are transplanted into the central nervous system do not display any migratory ability. Delivery of vector is further reduced as this procedure involves xenotransplantion of mouse cells into humans. These cells elicit an immune response which eventually leads to their elimination and hence cessation of vector delivery. It can be argued that the immune response to the packaging cells is limited, since most brain tumor patients undergo treatment with immune suppressants, the CNS is a somewhat immune-privileged site, and brain tumors themselves may also suppress the immune system. Even if these arguments have some biologic significance, the fact that the packaging cells lack the ability to migrate, limits the therapeutic strategy from the beginning, since it does not address one of the basic properties of brain tumorsxe2x80x94invasiveness. By using the vector system of the present invention, where transforming cells into retrovirus packaging cells is simple, fast, and efficient, it will be possible to investigate and implement the biologic properties that these cells need to deliver genes to tumor cells efficiently in vivo.
One could go as far as proposing that tumor cells should themselves be transformed into packaging cells in cell culture, and implanted back into the tumor cavity. This ex vivo strategy of transforming cells into packaging cells according to the present invention has much broader applications than just tumor therapy, and could be used to transform other primary cells into packaging cells. For example, one could use the vector system of the present invention, which takes advantage of migratory and organ homing properties of different cells, for gene delivery to tissues or organs where cell proliferation occurs in the adult or during development.
The most exciting property of the vector system of the present invention, however, is the ability to transform cells into packaging cells in vivo. This can be achieved in a single transduction step, which can be accomplished using liposomes, electroporation, molecular conjugates and DNA guns, or any other method to introduce DNA into cells in vivo or by infection using the vector""s ability to be packaged into HSV virions.
As discussed above, retrovirus vectors have been long recognized as useful tools for gene transfer in cell culture, but with limited applicability in vivo due to their characteristic low titers, i.e., infectious units/ml of virus stock, inability to transduce non-dividing cells, and finally their fragility, which precludes efficient concentration of viral stocks. However, retrovirus present a tremendous advantage in relation to other vectors with their ability to stably integrate into the host genome. For these reasons, retrovirus vectors have been used mostly in ex vivo protocols where target cells are grown in culture, and after being infected with the desired vector and selected, they are transplanted back into the individual. What the vector system of the present invention can accomplish is to transform cells in vivo into packaging cells which will deliver retrovirus vectors over an extended period of time, without being destroyed by the process of packaging, as occurs during production of most other vectors used for gene delivery.
While the development of vectors that can efficiently infect non-dividing cells and achieve very high titers has been accomplished by a series of vectors, including adenovirus, AAV, and HSV, there are still major hurdles that limit the therapeutic efficacy of those vectors. First, is the fact that the vectors that achieve the highest titers and infect non-dividing cells do not stably integrate into the genome and elicit strong immune responses from the host. Second, direct injection of the viral vector will only affect the organ or tissue in a very localized manner, not always enough to achieve a therapeutic effect. Several strategies have been developed to address this latter issue that make use of more generalized gene delivery protocols such as injection of vectors in the bloodstream, which in most cases, however, results in gene delivery to the liver. In the case of generalized gene delivery to the brain, several strategies have been tried such as the chemically induced temporary disruption of the blood brain barrier which allows vectors to cross it and infect cells in brain tumors or in the brain parenchyma. Although these strategies have been rather successful in gene marking experiments, the vectors used in those experiments have been unable to mediate stable gene expression for a variety of reasons that range from immune compromise of gene delivery to the episomal nature of those vectors.
Accordingly, the present invention overcomes the disadvantages of the prior art because the present hybrid amplicon vector systems take advantage of the migratory and organ or tissue homing properties of certain cells to deliver retrovirus vectors in situ by transforming those cells into retrovirus packaging cells. Furthermore, once the retrovirus vectors integrate into the genome of the target cells, this genetic element should be stable and transmitted to the progeny of those cells. Another property of this system is that it can be used both as an HSV-amplicon vector or as a plasmid to achieve the same goals.
Thus, the present invention provides hybrid amplicon vectors comprising genetic elements derived from several viruses: Herpes Simplex Virus (HSV), Epstein-Barr Virus (EBV) or Adeno-Associated Virus (AAV), and a retrovirus.
In one embodiment, the hybrid amplicon vector comprises elements from HSV, EBV, and a retrovirus. In a preferred embodiment, the hybrid amplicon vector comprises: (a) an HSV origin of replication (ori S); (b) an HSV packaging signal (pac); (c) an EBV origin of replication (ori P); (d) an expression cassette of the EBNA-1 protein of EBV; (e) gag, pol, and env genes of a retrovirus; and (f) a retroviral vector, containing at least one transgene of interest. Of course, other genetic elements may also be present in the amplicon portion of the construct, such as additional regulatory (i.e., promoter), therapeutic, reporter, or marker genes.
In a particularly preferred embodiment of the HSV/EBV/retrovirus vector embodiment, the hybrid vectors are HERE and HERA.
In another embodiment, the amplicon vector comprises elements from HSV, AAV, and a retrovirus. In a preferred embodiment, the hybrid amplicon vector comprises: (a) an HSV origin of replication (ori S); (b) an HSV packaging signal (pac); (c) an AAV rep gene; (d) an AAV ITR element; (e) gag, pol, and env genes of a retrovirus; and (f) a retroviral vector, containing at least one transgene of interest. Of course, other genetic elements may also be present in the amplicon portion of the construct, such as additional regulatory (i. e., promoter), therapeutic, reporter, or marker genes.
In a particularly preferred embodiment of the HSV/AAV/retrovirus vector embodiment, the hybrid vectors are HyRMOV Ampho and HyBPlacZ.
Both vector system embodiments are capable of generating retroviral packaging cells.
The retroviral vector will have at least one transgene inserted therein. The transgene(s) may be a reporter or marker gene, and/or a therapeutic gene. Representative examples of suitable reporter genes include: xcex2-galactosidase, green fluorescent protein (GFP), galactokinase, alkaline phosphatase, chloramphenicol acetlytransferase, luciferase, and xcex2-lactamase. Representative examples of suitable selectable marker genes include gene sequences capable of conferring host resistance to antibiotics (such as ampicillin, tetracycline, kanamycin, etc.), amino acid analogs, or genes permitting growth of bacteria on additional carbon sources or under otherwise impermissible culturing conditions. The therapeutic transgene sequence may be a gene sequence associated with diseases and disorders including, but not limited to, inherited metabolic disorders, including, lysosomal storage disease, Lesch-Nyhan syndrome, inherited neurological diseases, including, amyloid polyneuropathy, Alzheimer""s Disease, Duchenne""s muscular dystrophy, ALS, Parkinson""s Disease and brain tumors, diseases of the blood, such as, sickle-cell anemia, clotting disorders and thalassemias, cystic fibrosis, diabetes, disorders of the liver and lung, heart and vascular disease, diseases associated with hormone deficiencies, movement disorders, pain, stroke, cancer, and HIV.
The invention further provides a method for expressing a transgene in a proliferating cell population, in vitro or in vivo using the hybrid vectors of the invention. Some exemplary in vivo applications for the gene delivery system of the invention include gene delivery to the central nervous system during neurogenesis and gliogenesis, gene delivery to the bone marrow for the correction of genetic disorders as well as to protect the bone marrow from infection (as in HIV-infected individuals or other immune deficient individuals), gene delivery to the developing liver, and gene delivery to the lung during development.
The invention also provides a method of treating diseases and disorders using the hybrid vectors of the invention. Non-limiting examples of the diseases and disorders that can be treated using the present hybrid vectors include: inherited metabolic disorders, including, lysosomal storage disease, Lesch-Nyhan syndrome, inherited neurological diseases, including, amyloid polyneuropathy, Alzheimer""s Disease, Duchenne""s muscular dystrophy, ALS, Parkinson""s Disease, diseases of the blood, such as, sickle-cell anemia, clotting disorders and thalassemias, cystic fibrosis, diabetes, disorders of the liver and lung, heart and vascular disease, diseases associated with hormone deficiencies, movement disorders, pain, stroke, HIV, tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphoma, astrocytomas, oligodendrogliomas, meningiomas, neurofibromas, ependymomas, Schwannomas, neurofibrosarcomas, and glioblastomas.
In one preferred embodiment, the invention provides a method of selectively killing neoplastic cells using the hybrid vectors of the invention.
The invention also provides a preferred embodiment of the foregoing vector wherein any of the above mentioned hybrid amplicon vectors are capable of generating retrovirus packaging cells in a single step. One or two hybrid amplicons can be used to deliver the retroviral vector components.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.